Antimicrobial activity of some medicinal plants against some Gram

positive, Gram negative and fungi

By
Sanaa O. Yagoub 1; Shami El Haj Al Safi 1 ; Braaha Ahmed 1 and Asha Z. El Magbol 2
1.

Department of Microbiology and Molecular Biology , Faculty of Science and

Technology, El Neelain University , Sudan E.mail soyagoub@hotmail.com.

2.

Medicinal and Aromatic Plants, Ministry of Science and Technology, Khartoum,

Sudan.

Abstract

The petroleum ether , methanol and aqueous extracts of the seeds of Helianthus
annuus (Asteraceae) , leaves of Azadirachta indica (Meliaceae), bulbs of Allium cepa
(liliaceae) and that of the seeds of Portulaca oleracea (Portulacaceae) were screened for
their anti-microbial activity using the Cup plate agar diffusion method . They were tested
against six bacteria; two Gram-positive bacteria (Bacillus subtilis and Staphylococcus
1

aureus) and four Gram-negative bacteria (Escherichia coli, Proteus vulgaris, Pseudomonas
aeruginosa and Salmonella typhi) and against two fungi (Aspergillus niger and Candida
albicans).
The susceptibility of the microorganisms to the extracts of these plants was
compared with each other and with selected antibiotics. The antimicrobial activities of
these plants were discussed according to their phytochemical components.
Key words: Antimicrobial activity ,Gram positive ,Gram negative.fungi, Medicinal
plants, Helianthus annuus, Azadirachta indica, Portulaca oleracea,
Allium cepa. Allium cepa
Responsible author: Sanaa Osman Yagoub, E.mail soyagoub@hotmail.com.

Introduction
Azadirachta indica. Juss. Family name: Meliaceae ,Vernacular name:
Neem distributed widespread in the world. The Chemical constituents contain many
biologically active compounds that can be extracted from neem , including alkaloids,
flavonoids, triterpenoids , phenolic compounds , Carotenoids , steroids and ketones ,
azadirachtin

is

actually

mixture

of seven isomeric compounds labeled as

azadirachtin A-G and azadirachtin E is more effective (Verkerk et al., 1993) .Other
compounds than azadirachtin that have a biological activity are salannin** , volatile
oils, meliantriol and nimbin (Alghazali et al., 1998; Jacobson,1990 ; National Research
Council 1992).
Neem leaf is effective in treating eczema, ringworm, acne, anti-inflammatory,
antiheperglycemic and it is used to heal chronic wounds , diabetic foot and gangrene
developing conditions . It is believed to remove toxins from the body, neutralize free
radicals and purify the blood. It is also used in treatment of malaria. Recently it used as
anti-cancer and it has hepato-renal protective activity and hypolipidemic effects (Ahana,
2005). The juice of green neem leaves drunk with milk increase appetite, uses as a
collyrium, the juice instantly relieves headaches and cures eyes infections. Boiled neem
leaf water makes an excellent antiseptic to clean wounds, soothes, swellings and eases
skin problems (***).
Helianthus annuus,family name: Asteraceae (composite), Vernacular name:
Abbad al-shams, distributed worldwide. The plant contains an oleic acid and the new tri
acyl glycerol was identified as isomers of oleic acid from high-saturated sunflowers (Fern
2

et al., 2000). Also, it contains alkaloids, cyanogenic glycosides, saponins, cardiac

glycosides, tannins, fixed oils and simple phenolic (Alghazali et al.,1998) . It also
contains polysaccharides complex and rarely tocopherol or vitamin E- anti oxidant
(Dorrell, 1981) and (Elshami and Zen El-din 1991).
The Seeds are used medicinally as diuretic, expectorant and are used for cough,
throat and lung ailments. It is also used as antiseptic, aphrodisiac, deobstruent, emollient
and as anti-malaria. It is a folk remedy for blindness, bronchitis, carbuncles, catarrh,
colic, diarrhea, dysentery, dysuria, eyes, fever, inflammation, laryngitis, menorrhagia,
pleuritis, rheumatism, scorpion stings, snakebite, splenitis, urogenital ailments, whitlow
and wounds (Duki and Wain,1981). It is also used as anticancer (Hartwell., 1971).
Allium cepa L.Family name: Liliaceae ,Vernacular name: Basal. The onion is
Distributed World wide. The onion bulbs contains numerous organic sulfur compounds,
including trans-S-(1- propenyl) cysteine sulfoxide, Smethylcysteine sulfoxide, S
propylcysteine sulfoxide and cycloalliin; flavonoids; phenolic acids; sterols including
cholesterol, stigma sterol, b-sitosterol; saponins; sugars and a trace of volatile oil
composed mainly of sulfur compounds, including dipropyl disulfide (Kapoor, 1990;
Leung and Foster, 1996). A fresh onion bulb contains fructans with a low degree of
polymerization, and sulfur-containing compounds (Bruneton, 1995).
Onion was used for decrease cancer tumor initiated, promote healing of stomach
ulcers, inhibit the proliferation of cultured ovarian, breast and colon cancer cells; reduce
the cholesterol, blood pressure and symptoms associated with diabetes mellitus, inhibit
platelets aggregation (involved in thrombosis) and prevent inflammatory processes
associated with asthma (Dorsch and Wanger, 1991; Augusit, 1996). Onion was used as
antiseptic, antiheleminthic, antispasmodic, carminative, diuretic, cholagogge, diaphoretic
and expectorant. It was used also for coughs, the flu, parasites, wound, burns, dog bites,
bee stings, earaches, athletes' foot, warts, baldness, toothaches, intestinal infections,
kidney infections, contaminated blood and heart failure. Raw onion can completely
sterilize your mouth and throat (Mehrabian et al., 2005).
Portulaca oleracea Family name: Portulacaceae Vernacular name: (Rigla) Distributed
World wide. Portulaca oleracea contains many biologically active compounds and is a
source of many nutrients. Some of the biologically active include free oxalic acids, alkaloids,
Omega-3 fatty acids, coumarins, flavonoids, cardiac glycosides and anthraquinone
glycosides. It has high contents of Omega-3 fatty acids and proteins. Some of the compounds
in Portulaca oleracea are alanine, caffeic acid, calcium oxalate, catechol, beta-cyanine,
digalactosyldiacyl glycerol, docosahexaenoic acid, dope, eicosapentaenoic acid, HCN,
3

histidine, L-noradrenalin, linoleic acid, alpha-linoleic acid, lysine, menthionine, saponins, nor
epinephrine, oleic acid, oxalates, phytin-p, sinapic acid, tannin, beta-sitosterol, valine,
threonine, tryptophan and vitamins A & C (Ezekwe et al., 1999).Portulaca oleracea was
used as antidiarrhoeal, antihelminthic, antiphlogistic and bactericide in bacillary dysentery,
hemorrhoids, enterorrhagia, antidiabetic, externally used as cataplasm of fresh leaves for
maturing of abscesses, seeds used as calmative and Salk thirst, diuretic, refreshing agent,
antiscrbutic, emollient and vermifuge (Boulos,1983).It was used as wound healing and as
anti-inflammatory (Leung and Foster, 1996).
Materials and Methods
Plant materials:
The four plants used in this study were Azadirachta indica and Helianthus annuus ,
Allium cepa and Portulaca oleracea were collected from ALSAHAFA and KADOUS
(February 2005). The voucher of these plants was deposited at the herbarium of the
institute of medicinal and Aromatic plants, Ministry of Science and Technology.
The leaves of Azadirachta indica , seeds of Helianthus annuus and bulbs of Allium
cepa and Portulaca oleracea were air-dried, coarsely powdered and were then extracted.
Preparation of the crude extracts:
Hundred grams of each of the air-dried and coarsely powdered plant material was
exhaustively extracted for 2 hours with petroleum ether (60-80oC) in soxhlet apparatus.
The petroleum ether extract was filtered and evaporated under reduced pressure using
Rota-vapor (Heidolph, Heizbad, Laborota 4001,Germany, 2000 ). The extracted plant
material was then air-dried, repacked in the soxhlet apparatus and exhaustively extracted
with methanol (98.8%) for 2 hours. The methanol extract was filtered and evaporated
under reduced pressure using Rota-vapor.
The extracts were dissolved in dimethyl-sulphoxide to make the final
concentrations which kept in refrigerator till used.
Simultaneously, water extract was prepared by adding (10ml) of boiled distilled
water to 5gm of coarsely powdered plant, s leaves in a beaker on water bath with
occasional stirring for 4 hours. The aqueous extract was then filtered and rewashed with
small volume of boiled distilled water and added to the filtrate, which were then adjusted
to (5ml) volume and used immediately
Preparation of the tested organisms:
A) Preparation of standard bacterial suspensions:

The average number of viable ,Bacillus subtilis (NCTC 8236), Escherichia coli
(ATCC 25922), Proteus vulgaris (ATCC 6380), Pseudomonas aeruginosa ( ATCC
27853), Salmonella typhi (ATCC 0650), Staphylococcus aureus (NCTC 25953 organisms
per ml of the stock suspensions was determined by means of the surface viable counting
technique (Miles and Misra, 1938). About (108-109) colony-forming units per ml was
used. Each time, a fresh stock suspension was prepared; the experimental conditions were
maintained constant so that suspensions with very close viable counts would be obtained.
B) Preparation of standard fungal suspensions:
The fungal cultures (Aspergillus niger (ATCC 9763) , Candida albicans ( ATCC
7596) were maintained on Saboraud dextrose agar, incubated at 25C for 4days. The
fungal growth was harvested and washed with sterile normal saline and finally suspended
in (100ml) of sterile normal saline and the suspension was stored in refrigerator till used.
In vitro testing of extracts for antimicrobial activity:
Testing for antibacterial activity:
The cup-plate agar diffusion method was adopted according to Kavanagh, (1972) to
assess the antibacterial activity of the prepared extracts. 0.6 ml of standardized bacterial
stock suspensions (108-109) colony- forming units per ml was thoroughly mixed with 60
ml of sterile nutrient agar. 20 ml of the inoculated nutrient agar were distributed into
sterile Petri dishes. The agar was left to set and in each of these plates 4 cups, 10 mm in
diameter, were cut using a sterile cork borer No. 4 and the agar discs were removed.
Alternate cups were filled with 0.1ml of each extracts using microtiter-pipette and
allowed to diffuse at room temperature for two hours. The plates were then incubated in
the upright position at 37C for 18 hours. Two replicates were carried out for each extract
against each of the test organism. Simultaneously addition of the respective solvents
instead of extracts was carried out as controls. After incubation the diameters of the
results and growth inhibition zones were measured, averaged and the mean values were
tabulated.
Testing for anti-fungal activity:
The same method as for bacteria was adopted. Instead of nutrient agar, yeast and
mould extract agar was used. The inoculated medium was incubated at 25C for two days
for the Candida albicans and three days for Aspergillus niger.
Results
The petroleum ether and aqueous extract of seeds of Helianthus annuus , bulbs of
Allium cepa, Portulaca oleracea and leaves of Cassia senna were found to be inactive
5

against all organisms tested. The methanol extract showed different antimicrobial activity
toward test organisms. The methanol extract of seeds of Helianthus annuus showed low
activity (14mm) against Bacillus subtilis, high activity (23mm) against Proteus vulgaris
and inactive against the rest of tested organisms. All extracts of Helianthus annuus were
inactive against Aspergillus niger and Candida albicans. The methanol extract of bulbs
of Allium cepa exhibited high activity against Bacillus subtilis (23mm), Proteus vulgaris
(20mm) , Pseudomonas aeruginosa (23mm) and inactive against Staphylococcus aureus,
Escherichia coli, Salmonella typhi and against Aspergillus niger and Candida albicans .
The methanol extract of the leaves of Azadirachta indica exhibited pronounced activity
(28mm) against Bacillus subtilis, high activity (18mm) against the Gram-positive
Staphylococcus aureus and the Gram-negative organisms Proteus vulgaris, (20mm)
against Salmonella typhi , low activity (14mm) against Pseudomonas aeruginosa and
inactive against Escherichia coli . All extracts were inactive against Aspergillus niger.
Both petroleum ether and methanol extracts o f the leaves of Azadirachta indica showed
high activity (15-18mm) against Candida albicans, while its aqueous extract was
inactive. The methanol extract of Portulaca oleracea showed high activity against both
Gram-positive organisms, Bacillus subtilis (20mm) , Staphylococcus aureus (15mm) and
against one Gram-negative bacterium Pseudomonas aeruginosa (18mm) , low activity
against Candida albicans (14mm) and inactive against Escherichia coli , Proteus
vulgaris , Salmonella typhi and Aspergillus niger
The seeds methanol extract of Helianthus annuus was found to be as effective as
100g/ml Gentamicin against Proteus vulgaris. The leaves methanol extract of
Azadirachta indica was found as effective as 100g/ml Gentamicin against Bacillus
subtilis and 20g/ml Tetracycline against both

Staphylococcus aureus and Proteus

vulgaris and 10g/ml Gentamycin against Pseudomonas aeruginosa. The bulbs methanol
extract of Allium cepa at 100mg/ml concentration was found to be effective similar to that
of 20g/ml Tetracycline against Bacillus subtilis, 40g/ml Gentamicin against Proteus
vulgaris and 100g/ml Gentamicin against Pseudomonas aeruginosa. The methanol extract
of seeds of Portulaca oleracea at 100mg/ml concentration was effective as 20g/ml ,
10g/ml Gentamicin and 10g/ml Tetracycline against
Gentamicin

against

Staphylococcus aureus

Pseudomonas aeruginosa.

and

Bacillus subtilis ,

20g/ml

Gentamicin

10g/ml
against

Table (1): Preliminary screening for antimicrobial activity of different plants against
standard organisms:
Micro
organisms

Bacillus
subtilis
Staph.
aureus
E.coli
Proteus
vulgaris
Pseudo
aeruginosa
Salmonella
typhi
Aspirogillus
niger
Candida
albicans

Mean Diameter Inhibition Zone(mm)

Helianthus anuusns
Azadirachta indica
Allum cepa
Portulac
P.
Methanol Water P.
Methanol Water P.
Methanol Water P.
Met
ether
ether
ether
ether
(-)

14

(-)

(-)

28

(-)

(-)

23

(-)

(-)

(-)

(-)

(-)

(-)

18

(-)

(-)

(-)

(-)

(-)

(-)
(-)

(-)
23

(-)
(-)

(-)
(-)

(-)
18

(-)
(-)

(-)
(-)

(-)
20

(-)
(-)

(-)
(-)

(-)

(-)

(-)

(-)

14

(-)

(-)

23

(-)

(-)

(-)

(-)

(-)

(-)

20

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(18)

15

(-)

(-)

(-)

(-)

(-)

Table (2): Screening of antibacterial activity of Gentamicin and Tetracycline against

standard organisms:
Conc.
Drug

Gentamicin

Tetracycline

MDIZ(mm)

g/ml

B.s

S.a

E.coli

Pr.v

Ps.a

Sa.t

100

30

20

25

22

25

40

30

18

24

20

20

20

20

17

23

17

15

10

20

14

20

15

14

100

27

31

24

40

25

30

25

20

24

26

23

10

20

22

20

MDIZ: Mean diameter of growth inhibition zone in (mm).

B.s: Bacillus subtilis

S.a: Staphylococcus aureus
E.coli: Escherichia coli
Pr.v: Proteus vulgaris
Ps.a: Pseudomonas aeruginosa
Sa.t: Salmonella typhi
-: No inhibition zone

Table (3): Screening of antifungal activity of Nystatin against standard organisms:

8

Drug

Conc. g/ml

Nystatin

MDIZ(mm)
A. niger

C.alb

50

17

28

25

14

26

12.5

23

MDIZ: Mean diameter of growth inhibition zone in (mm).

*: Average of two replicates.
A.niger: Aspergillus niger
C.alb: Candida albicans
-: No inhibition zone

Discussion
The

petroleum ether, methanol and aqueous extracts of

Helianthus annuus (Asteraceae) and Portulaca oleracea ,

the

seeds of

the leaves of Azadirachta

indica (Meliaceae) and bulbs of Allium cepa (liliaceae) were subjected to a preliminary
screening for antimicrobial activity against six standard bacteria ( Bacillus subtilis,
Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa
and Salmonella typhi ) and two fungi (Aspergillus niger and Candida albicans). It was
clear from table (1), that both petroleum ether and aqueous extracts of the seeds of
9

Helianthus annuus , seeds of Portulaca oleracea, leaves of Azadirachta indica and bulbs
of Allium cepa showed no activity against all organisms tested, unlike its methanol
extracts. Methanol extract of Helianthus annuus showed low activity against Bacillus
subtilis (14mm), pronounced activity against Proteus vulgaris (23mm) and inactivity
against the rest of the organisms tested these antibacterial activity might supported by
Alghazali et al.,1998 who mentioned that this plant chemically include alkaloids ,
cyanogenic glycosides , saponins , cardiac glycosides , tannins , fixed oils and simple
phenol compounds. All extracts were inactive against Aspergillus niger and Candida
albicans.
The methanol extract of Azadirachta indica exhibited pronounced activity against
Bacillus subtilis (28 mm), high activity against the Gram-positive organism
Staphylococcus aureus (18mm), the Gram-negative bacteria Proteus vulgaris (18 mm)
and Salmonella typhi (20 mm), low activity against Pseudomonas aeruginosa (14 mm)
and inactive against Escherichia coli . These might be due to presence of triterpenoids ,
phenolic compounds ,

Carotenoids , steroids , valavinoids, ketones and tetra-

triterpenoids azadirachtin These results were similar to those reported by Ikram and
Inamul 1980a, 1980b. All extracts were inactive against Aspergillus niger. The
petroleum ether and methanolic extracts of Azadirachta indica exhibited high activity
against Candida albicans (15-18mm); while its aqueous extract was inactive against
Candida albicans.
The methanolic extract of bulbs of Allium cepa showed pronounced activity (23mm)
against Bacillus subtilis and Pseudomonas aeruginosa, high activity (20mm) against
Proteus vulgaris, while inactive against Staphylococcus aureus, Escherichia coli and
Salmonella typhi. The onion bulbs contains numerous organic sulfur compounds,
including trans-S-(1- propenyl) cysteine sulfoxide, Smethylcysteine sulfoxide, S
propylcysteine sulfoxide and cycloalliin; flavonoids; phenolic acids; sterols including
cholesterol, stigma sterol, b-sitosterol; saponins; sugars and a trace of volatile oil
composed mainly of sulfur compounds. The presence of these compounds may explain
the antimicrobial activity of these plants. These results were different from that produced
by Merih and Buket, 1997; who reported that the onion inhibited the growth of
Salmonella typhi and Escherichia coli. Although our three extracts did not show any
activity against Staphylococcus aureus; the study done by Merih and Buket, 1997;
Elnima et al., 1983 and Noureddine et al., 2005 showed some sensitivity.

10

Extracts of both bulbs of Allium cepa and Portulaca oleracea were inactive against
both Aspergillus niger and Candida albicans. Noureddine et al., 2005 showed that
Aspergillus niger was significantly inhibited at high concentration of these two plants
extracts. Kabelik, 1970 found that Allium cepa was found to be active against Candida
albicans, although our extract showed same degree of antibacterial activity, but not
antifungal activity. Merih and Buket, 1997; Hughes and Lawson, 1991 and Augusit,
1996 reported that onion extracts have both antibacterial and antifungal properties.
Methanol extract of Portulaca oleracea showed high activity against both Gram-positive
organisms Bacillus subtilis (20mm) and Staphylococcus aureus (15mm) and only active
against one Gram-negative bacteria namely Pseudomonas aeruginosa (18mm) These
might refer to the presence of coumarins, flavonoids and saponins as chemical
components of these plant . Shahidi et al., 2005, determined similar results and found
that the methanol extract of Portulaca oleracea seeds (20mg/ml) was active against
Staphylococcus aureus, Bordetella bronchiseptica and Bacillus cereus.
All extracts of the seeds of Portulaca oleracea were inactive against Aspergillus
niger. The methanol extract of Portulaca oleracea exhibited low activity against Candida
albicans. Bongoh et al., 2000 showed that the extract of Portulaca oleracea showed
antifungal properties against Aspergillus niger and Candida albicans.
It is not surprisingly that there are difference in the antibacterial activities of
the extracts of the different extracts of tested plants This could be due to the
phytochemical differences between them.
References

Ahana Nagda (2005). The medicinal value of Azadirachta indica. Hindu Press,
India.
Aizenman, B.E. (1978). Higher plants as a source for the preparation of new
antibiotics. Microbial. Zh (Kiev) 40, 23.
Augusit, K. (1996). Therapeutic values of onion and garlic. Indian Journal of
Experimental Biology. 34: 634-640.
Bongoh; Moochang and Jun Hwang (2000). Detection of antifungal activity in
Portulaca oleracea by a single cell bioassay system Phytotherapy research, vol.14,
Issue 5, 329-332.
11

Bruneton, J. (1995). Pharmacognosy, Phytochemistry, Medicinal Plants Pairs;

Lavoisier Publishing.
Dorell, D.G. (1981). Sunflower Helianthus annuus. In: McClure, T.A.and
Lipinsky, E.S. (eds), CRC hand book of biosolar resources. Vol.11. Resource materials
- CRC press, Inc., Boca Raton, fl. p.105-114.
Dorsch, W. and Wanger, H. (1991). New antiasthmatic drugs from traditional
medicine. Int Arch Allergy Appl Immunal. 94: 262-265.
Elghazali, E.B. Gamal; Mahgoub. Eltohami; Awatif, A.B. Elegami; Wail
Sadig Abdullah; Galal, M.M. (1997). Medicinal plants of Northern Kordofan Part IV,
National Center for Research, Medicinal and Aromatic Plants Research Institute
(MAPRI). Khartoum. p. 21.
El-nima, E.I.; Ahmed, S.A.; Mekkawi, A.G.

And Mossa, J.S. (1983). The

antimicrobial activity of Garlic and Onion extracts Pharmazie ; 38 (11) : 747-748

.(Internet search)
Elshami; Zen El Din, E.E. (1991). Separation and characterization of the
polysaccharide complex of Helianthus annuus and its application for the preparation of
sustained release dosage forms. Alazhar J.of Nat.prod, vol 7., pp.77-92.
Ezekwe; Michael; Omara; Alwala Thomas R. and Membrahtue Tedesse (1999).
Nutritive characterization of purulence accessions as influenced by planting date,
Plant Foods for human nutrition (Dordrecht), 54 (3): 183-191.
Hartwell, J.L. (1971). Plants used against cancer. A survey.

L loydia. 30-34.

Venezuela.
Ikram, M. and Inamual, H. (1980.a). Screening of Medical Plants For
Antimicrobial Activity. Part 1.Fitoterapia 51:231.
Ikram, M. and Inamual, H. (1980.b). Screening of Medical Plants For
Antimicrobial Activity. Part 2. Fitoterapia 51:281.
Jacobson, M. (1990). Review of neem research in the United States.

In: Locke,

J.C. and Lawson, R.H. (eds) proceedings of a workshop in neems potential in pest
management program. USDA-ARS. Beltsville, MD.ARS-86.PP.4-14.
12

Kabelik, J. (1970). Antimikrobielle Eigen's chaften des Knob Lauchs pharmazie,

25:266.
Kapoor, L.D. (1990). Handbook of Ayurvedic Medicinal Plants. Boca Raton; CRC
Press, 25.
Kavanagh, F. (1972). Analytical Microbiology. F. Kavanagh (ED), VOLII,
Academic press, New York, and London, P: 11.
Leung, A.Y. and Foster, S. (1996). Encyclopedia of Common Natural Ingredients
used in Food, Drugs and Cosmetic, 2nd Ed. New York, John Wiley & Sons, Inc.
Mehrabian, S. and Larry, H.-Yazdy (2005). Antimicrobial activity of Allium
sativum, Allium cepa, Allium porrum against entricpathogenes (Enterobacteracea).
(Internet search).
Miles, A.A. and Misra, S.S. (1938). Estimation of bacterial power of blood J.Hyg.
38:732.
National Research Council. (1992). Neem: tree for solving global problems.
National Academy Press, Washington, D.C.
Noureddine Benkeblia;

Said Dahmouni; Shuichi Onodera

and Norio Shiomi

(2005). Antimicrobial activity of Phenolic Compound Extracts of Various Onions

(Allium cepa L.) Cultivars and Garlic (Allium sativum L.) Journal of Food
Technology, 3 (1): 35-40.
Shahidi Bonjar, G.H.; Aghighi, S. and Kamiri Nik, A. (2004)***. Antimicrobial
and Antifungal Survey in Plants used in Endogenous Herbal-Medicine of South East
Regions of Iran. Journal of Biological Sciences 4 (3): 405-412.
Verkerk, R.H.J. and Wright, D.J. (1993). Biological activity of neem seed
kernel extract and synthetic azadirachtin against larvae of Plutella xylostellal. Pesticide
science, vol. 37: PP.83-91.

13

14